In a flow cytometer, particles of interest (i.e. cells, beads, or other microscopic objects) are transported in a carrier fluid though a cuvette or flow cell. As is well known in the art, some of these particles may be non-fluorescent whereas others may be marked with fluorescent labels that can be used to identify specific particle characteristics or may hold an inherent autofluorescence characteristic which, when excited, can cause emission of electromagnetic radiation (such as photon irradiation or emission).
Lasers are used to excite said labels or particles and signal detection by sensing devices allows parameters such as size, shape, DNA content, surface receptors, enzyme activity, membrane permeability and calcium flux, to name a few applications, to be measured. The present invention is used for signal (where the term “signal” can be considered to pertain to radiation contained within one or more of the UV, visible, or IR regions of the electromagnetic spectrum) collection. The objective lens is typically designed to gather light (scattered, fluorescent, or other) from particles flowing through the interrogation/observation region of the flow cytometer and produce an image that is magnified with respect to the original object. This magnified image may be spatially separated and carried on to be detected by one or more suitable sensing devices such as photomultiplier tubes as those familiar with this technology would appreciate.
Historically, flow cytometers have utilized microscope grade objectives because of the common availability of stock lens parts and designs. Since microscopes are vision systems the optical aberrations are designed to be very low. This insures good quality so that the image can be viewed clearly by the human eye. However, flow cytometers are typically not vision systems and the same amount of image quality has not previously been required to achieve adequate results on a cytometer. The focused image created by the cytometer objective is often formed for simple optical path separation since there may be multiple lasers acting on the stream of particles at one time. It is necessary to maintain a level of quality in the image in order to proceed down the correct respective path and maintain the signal/photons to be detected by the sensing devices. However, this is a lesser level of image quality than what is required when the sensing device is the human eye as is the case in many microscopes.
Specialized optical collection lens systems for flow cytometry that do not rely on commercial microscope objectives have been described. One such prior-art lens system 100 for use in flow cytometry is illustrated in FIG. 1. The lens system 100 (FIG. 1) comprises a transparent plate 108, a plano-convex lens 110 optically coupled to the plate 108, a first meniscus lens 112 optically coupled to the plano-convex lens 110 at a side opposite to the plate 108 and a second meniscus lens 114 optically coupled to the first meniscus lens 112 opposite to the plano-convex lens 110. The prior-art system 100 further comprises a first doublet lens 116 optically coupled to the second meniscus lens 114 at a side opposite to the first meniscus lens 112 and a second doublet lens 122 optically coupled to the first doublet lens 116 as a side opposite to the second meniscus lens 114.
The lens system 100 (FIG. 1) is adapted to magnify the image of and collect light emitted or scattered from an object (OBJ) 102 (typically, biological cellular material) situated within a solution (typically saline water) passing through or housed within a cuvette comprising cuvette walls 104. An optical gel layer 106 provides an interface between the cytometry flow cell and the lens proper and improves lens mounting tolerances.
The plano-convex lens 110 of the prior-art system 100 (FIG. 1) is of a single material of less than 1.55 refractive index and has a planar surface defining an object side of the system and a convex surface having a radius of curvature in a range from 3.5 to 5.5 mm. The two meniscus lenses have concave surfaces facing the object side of the system, the surfaces of the second meniscus lens 114 being less sharply curved than corresponding surfaces of the first meniscus lens 112 and the convex surface of the first meniscus lens 112 being less sharply curved than the convex surface of the plano-convex lens 110.
Although the prior-art flow cytometry lens system 100 is adequate for its intended purposes, it has an overall track length (object to image distance) of over 176 mm and produces a geometrical spot size of 85.04 μm at full field and 71.86 μm on-axis, thereby putting a minimum of 80% of the optical energy of the image of an infinitely small point source within a circle of less than 200 μm diameter. Although a loss in image quality is acceptable for a flow cytometer, it is desirable to have an improved level of resolution for improved signal delivery, optical path separation and spectral resolution. Also, it is desirable to maintain the track length and the lens length (total thickness of all lenses along an optical axis) as small as possible, since space conservation and weight minimization are important considerations in the construction modern flow cytometers. Therefore, the present invention is aimed at improving the resolution and track length relative to the prior art while maintaining a suitable level of chromatic performance. This will allow for greater photon collection and ensure proper delivery of signal to a photo-detection block (comprising a plurality of sensing devices). In particular, a 25% reduction in on-axis and full field RMS spot size is desired with an 80% energy containment radius of 100 μm or less. Numerical aperture should be maximized considering a square cuvette channel to be no less than 0.94. Primary and secondary axial color aberrations should have absolute values less than 0.2 mm.
In addition, the present invention is aimed at reducing the size of the flow cytometer by reducing the overall collection optic track length (as compared to the prior art) by 33%, reducing the lens barrel diameter by over 50% and slightly reducing the lens length. It is desirable to keep the collection optic track length and lens length at less than or equal to 118 mm and 35 mm, respectively, since space and weight conservation are important considerations in the design and construction of today's flow cytometers. In short, the present invention should be physically small with improved resolution and greater chromatic performance as compared to the prior art but should utilize the fact that a cytometer does not need vision quality optics.